Topical cosmetic treatment of skin and scalp and corresponding active ingredient based on an extract of Apium graveolens

ABSTRACT

The invention provides the use of at least one alkyl-phthalide or a plant extract comprising mainly said alkyl-phthalide for the topical cosmetic treatment of the skin and of the scalp, in particular an anti-dandruff treatment. According to the invention, a CO2 supercritical extract of Apium graveolens seeds consisting mainly of sedanenolide may be used.

This application is the U.S. National Phase application of PCTInternational Application No. PCT/IB2016/051760, filed Mar. 29, 2016,which claims the benefit of priority of French Patent Application No. FR1552692, filed Mar. 30, 2015, the contents of which are incorporatedherein by reference in their entirety for all purposes.

TECHNICAL FIELD

The present invention relates to a novel topical cosmetic treatment ofthe skin and scalp and a cosmetic active ingredient for said treatmentbased on an extract of Apium graveolens.

Cosmetics, hygiene and personal care, and dermo-pharmacy industries areconcerned.

BACKGROUND ART

One of the challenges of cosmetics is to fight strongly againstdandruff, often a benign condition of the scalp, but aestheticallyunpleasant. This condition is characterized by the presence of deadcells or whitish scales on the scalp, in hair and on clothing, oftenaccompanied by itching.

The origin of dandruff is related to the disturbance of one or severalfactors. As such, the increased production of sebum, which begins inadolescence, is an important factor although not sufficient. This sebumserves as substrate for yeast of Malassezia type, said yeast multiplyingthemself and producing irritating lipids disrupting the balance(homeostasis) of the scalp. Having sebum, yeast or a dry scalp is notenough to have dandruff. Individual susceptibility and external factorsplay an important role also. Among these are found in particular: hairtreatments that are too aggressive, extreme weather conditions, poordiet, fatigue, stress or pollution.

Two types of dandruff can be distinguished. In the dry form of dandruff,scalp seems to be too dry, the amount of dandruff is often low tomoderate; scales are small and isolated. Mild itching is often, althoughscalp is not red. This condition can evolve into a form represented byoily dandruff although the latter may appear immediately associated withan abundant production of sebum. Scales are thicker, larger andassociated with each other to form fatty, wet and yellowish plaques.Thereafter the scalp has often inflammatory changes, with more or lessspreading redness, irritating condition and itching.

The present invention aims in particular to provide a means forpreventing or treating unsightly dandruff and signs that accompany themsuch an oily and shiny skin, irritation and itching.

The Apium graveolens plant, commonly called celery, is part of thefamily of Apiaceae or Umbelliferae family, which includes many otheredible plants such as carrot and coriander. This plant is not toxic; allparts of it can be consumed as raw or cooked vegetables. The seeds arealso used as spices.

Italy is the country of origin, this plant being widespread in theworld, especially in Europe, Egypt, Algeria, Ethiopia, Asia and India.

Celery seeds contain about 3% of volatile oil and 15% of non-volatilevegetable oil.

An essential oil extracted from celery seeds comprises terpenes(D-limonene>60% and selinene 10-20%) and phthalides (1-4%). Among thepredominant phthalides, there is the 3-n-butyl-phthalide, the sedanolideand sedanenolide (also called senkyunenolide A).

The nonvolatile part includes petroselinic, oleic, linoleic, myristic,palmitic, palmitoleic, stearic and myristoleic fatty acids.

The Apium graveolens therefore comprises potentially very many activecompounds. As a medicinal plant, it is recommended for thousands ofyears for many properties: aphrodisiac, anthelmintic, antispasmodic,anti-inflammatory, carminative, diuretic, emmenagogue, laxative,sedative, stimulant and tonic. The plant can be used against asthma,bronchitis and rheumatism. The wild celery seeds are used in traditionalmedicine in India and other countries that have adopted the Ayurvedictraditions to heal, as a tranquilizer, antispasmodic, nerve tonic,diuretic and antirheumatic. The phthalides included in the Apiumgraveolens are described as active against cancer, high blood pressureand cholesterol. The world of living organisms and plants in particularrepresents a very important source of innovative active molecules forcosmetics and pharmaceuticals. To extract and concentrate them, thereare different methods, and the composition of the obtained extract willobviously depend on the method and the operating conditions.

The extraction technique with supercritical CO₂ is already widely usedin the food, pharmaceutical and perfumery industries. Gaseous CO₂,present in low concentrations in the air, is compressed so that itbecomes liquid. It is then an excellent solvent for fat-solublemolecules. Inert, it reacts little with the extracted molecules andleaves no residue since it is evaporated at the end of process andcaptured to be reused.

An essential oil of celery, obtained by hydro-distillation, comprisingin majority D-limonene has been proposed in cosmetics to treatpigmentation spots.

SUMMARY OF THE INVENTION

The present invention provides the use of at least one alkyl-phthalideor a plant extract comprising said alkyl-phthalide as major compound,for the non-therapeutic cosmetic topical treatment of the skin or scalp.

“Cosmetic treatment” means a treatment that addresses a normal, healthyskin or scalp, said treatment being intended to improve the appearanceand condition in order to beautify and/or reduce of any feelings ofdiscomfort. Such treatment has no therapeutic aim.

“Alkyl-phthalide” encompasses according to the invention phthalides inwhich the 6-carbon ring is aromatic and hydro-phthalides in which the6-carbon ring has only 1 or 2 unsaturated bonds.

An alkyl-phthalide according to the invention has therefore thefollowing general formula I:

R being an alkyl chain of 1 to 8 carbon atoms, preferably 1 to 4, andpreferably 4 (butyl phthalides), linear or branched, that can besubstituted by an OH or amine function (secondary or tertiary).

The present invention encompasses pure alkyl-phthalides which can beobtained by chemical synthesis or by extraction and purification from aplant comprising them.

It also encompasses plant extracts, such as Apium graveolens, which arecharacterized in that they predominantly comprise at least onealkyl-phthalide as active compound.

In vitro and in vivo tests detailed thereafter in the description showthe effects of the cosmetic treatment according to the invention,especially on the scalp, for preventing and/or treating dandruffconditions, to improve the mechanical properties of the dermalextracellular matrix (density, firmness, fine lines and wrinkles inparticular) and to prevent and/or treat an oily skin.

Specifically, it has been shown that the cosmetic treatment according tothe present invention is capable of acting at 5 levels on the scalpand/or skin:

-   -   The skin microflora;    -   The amount of sebum;    -   The skin barrier;    -   The cutaneous irritation/itching; and    -   The extracellular matrix of the dermis.

1) Regarding the Skin Microflora

Yeast of Malassezia type are present on almost every scalp at the levelof the hair follicles. Their presence in itself is not a problem in manypeople without dandruff, but their proportion in the skin microflora iscorrelated with dandruff state.

They secrete specific phospholipases and lipases whose role is toextract energy from triglycerides of sebum or phospholipids. This inreturn forms lipidic byproducts, such as arachidonic acid or oleic acid,which can be irritating if present in too large amounts. In addition,some yeast belonging to the Malassezia genus, among which Malasseziafurfur and Malassezia globosa, cause interleukin-8 formation bykeratinocytes and induce hyperproliferation of these cells with as aconsequence an alteration of the skin barrier function (stratumcorneum), which promotes even more the negative action of theby-products of bacteria and yeast such as oleic or arachidonic acids andmakes the scalp more sensitive. Improving the condition of the scalpthus requires control of the yeast of the Malassezia genus that cancause major disruptions.

In vitro and in vivo tests given below show that according to theinvention, these microorganisms and dandruff are fight.

2) Regarding the Sebum Production

Sebum is a mixture of complex lipids (triglycerides, fatty acids, waxes,esterified sterols, cholesterol and derivatives, and squalene) producedby sebocytes. From a physiological point of view, sebum is intended toprotect the skin by isolating it from the outside. It is clearly knownnow that it plays a role in the emergence of dandruff states. Indeed,the overproduction of sebum at puberty and incidence of dandruff statesincrease in parallel of one another. Moreover, dandruffs are seen onlyon areas rich in sebum.

As noticed above, these lipids are used as substrate for Malasseziayeast involved in dandruff states. Furthermore, as regard oily dandruff,a greater seborrhea accompanies overproduction of immature cells, gluesand maintains these on the scalp and hair.

Regarding the so-called oily skins, too much sebum will give them ashiny appearance, clog and open the pores, cause bacterial growth andcause skin imperfections including redness.

It is therefore necessary either for dandruff or oily skin to reduce theproduction of lipids by the sebocytes producing sebum.

3) At the Level of the Cutaneous Barrier

Skin and scalp are epithelia that form the stratum corneum. They arerenewed continuously by desquamation. This forms a very effectivebarrier with regard to external aggression and limits the water loss ofthe body. This barrier is an assembly of great complexity involving onone hand the cells having no nucleus, flat and strongly bonded togetherand, on the other hand, lipids whose composition and assembly providethe unique properties of this structure very resistant to physical,chemical and biological environmental aggressions.

In healthy skin, the stratum corneum is formed by the terminalmaturation of viable underlying cells of the granular layer. Superficialviable cells of the epidermis, having a lipid contour, are transformedinto little flat bricks coreless, the corneocytes, thanks in particularto proteins, such as involucrin, loricrin, filaggrin and SPRRs (smallproline-rich region proteins), all linked together. The SPRRs operate asbridging agents strengthening the horny envelope and governing thetoughness, strength and flexibility properties. Other proteins areexpressed as the LCE3B (Late Cornified Envelope protein 3B) induced whenit is necessary to repair the skin barrier. The corneocytes are stronglylinked together by corneodesmosomes and “cemented” by organized layersof extracellular complex lipids: cholesterol, ceramides and neutrallipids.

An optimal skin barrier function ensures that, thanks to desquamation,skin and scalp eliminate their superficial cells in an insensitive andnot visible manner. Desquamation is permitted by serine proteases, theKLK (Kallikrein-related peptidase), which break the ties of thecorneocytes between each other's (corneodesmosomes), the SPINK5 (SerineProtease Inhibitor Kazal-type 5) regulating their activity. Loss offunction of the SPINK5 causes hyper-peeling of immature cells and lossof homeostasis of the stratum corneum.

The loss of integrity of the skin barrier is often observed in dandruffstates. The division of the cells of the basal layer of the epidermisbecomes too fast. The cells that rise to the surface of the skin do nothave time to finish their maturation and develop into small corneocyte.Instead, larger cell structures are formed and therefore more visible,having a core residual (dandruff). This abnormal acceleration ofdesquamation facilitates the penetration of molecules and externalagents that can irritate the skin. This results in a disturbance of theprotective and regulatory functions of the stratum in particular theantimicrobial function.

In vitro and in vivo tests given below in the description show that,according to the invention, the skin barrier is reinforced, bystimulating the synthesis of proteins and lipids thereof, as well as thefunctional proteins LCE3B and SPINK5.

4) At the Level of Itching and Irritation of Skin

The disturbance of the scalp and dandruff states caused by a deficientskin barrier, the presence of Malassezia genus yeast in large amountsand/or any excess of sebum induce at the end itching and irritation forthe person. Red patches may even appear.

In vitro and in vivo tests given below in the description show that,according to the invention, skin itching and irritation are decreased.

5) At the Level of the Dermis Matrix

The loss of density and thickness of the dermis are in particularrelated to a reduction of the synthesis of collagen I by the dermalfibroblasts during cutaneous aging. Collagen I, being the most abundantprotein of the dermal extracellular matrix, is essential for firm andless wrinkled skin.

As a complement of activity, it has been shown that thealkyl-phthalides, or an extract of Apium graveolens seeds comprisingthem, according to the invention has the property of stimulating theproduction of collagen in dermal fibroblasts.

More generally, the present invention thus provides the use ofalkyl-phthalide(s) or a plant extract containing alkyl-phthalide(s) asmajor compound(s) for the following non-topical therapeutic cosmetictreatment(s):

-   -   Anti dandruff; and/or    -   Of irritation and/or itching of skin and scalp; and/or    -   To limit the production of sebum of skin and scalp; and/or    -   Of oily skin and/or dilated pores; and/or    -   To strengthen the skin barrier to restore skin homeostasis;        and/or    -   Anti-wrinkle and fine lines and to improve the mechanical        properties of the skin (loss of density, and thus loss for        example of firmness) through stimulation of collagen synthesis.

Most preferably according to the invention, used alkyl-phthalidescomprise sedanenolide, sedanolide and/or 3-n-butylphthalide. These threealkyl-phthalides, shown thereafter, are those comprised in the volatileportion of the seeds of the Apium graveolens plant.

Thus, according to a preferred feature of the invention, an extract ofApium graveolens seeds comprising sedanenolide, sedanolide and3-n-butylphthalide is used according to the invention. Other plants maybe used to extract one alkyl-phthalide according to the invention orproduce an extract containing it predominantly, particularly preferablyone of the 3 alkyl-phthalides present in the seeds of Apium graveolens.

And according to further preferred features according to the inventionan extract prepared by CO₂ supercritical extraction is used whichprovides an extract comprising alkyl-phthalides as major compounds,preferably at least 50%, preferably at least 65%, the remainingcompounds mainly consisting of terpenes and traces of fatty acids.

According to the invention the weight contents of these 3 phthalidesbased on total phthalides vary between:

Sedanolide: 0-45%

Sedanenolide: 45-90%

3-n-butylphthalide: 0-30%

Advantageously, an extract comprising preferably among the 3alkyl-phthalides the sedanenolide as the major compound, preferablycomprising at least 50%, and preferably at least 60%, and morepreferably at least 70% of sedanenolide based on total phthalides, isused.

The extract may be prepared by CO₂ supercritical extraction in apressure range of 75 to 300 bars and in a temperature range of 30° C. to80° C. Preferably the extraction will be done under 90 bars pressure andat 40° C.

Other extraction methods can be used to obtain an extract according tothe invention comprising a majority of at least one alkyl-phthalide,including extraction with a nonpolar solvent such as hexane.

The present invention also provides a cosmetic active ingredientcomprising in a physiologically acceptable matrix an oily extractcomprising mainly alkyl phthalides, said extract being obtainable bysupercritical CO₂ extraction of Apium graveolens seeds, and a cosmeticcomposition comprising at least said active ingredient in aphysiologically acceptable excipient.

DETAILED DESCRIPTION

The present invention will be better understood and other advantageswill appear from the following detailed description of preparationexamples and in vitro and in vivo tests.

Preparation of Compositions According to the Invention

A cosmetic composition, especially topical, comprises at least onealkyl-phthalide or a plant extract comprising in majority said at leastone alkyl-phthalide in a physiologically acceptable medium. According tothe excipient and the alkyl-phthalide dosage, this composition will be aconcentrated active ingredient or a final composition less concentrateddirectly for the end user.

“Physiologically acceptable medium” means according to the presentinvention, without limitation, an aqueous or hydro-alcoholic solution, awater-in-oil emulsion, an oil-in-water emulsion, a micro-emulsion, anaqueous gel, an anhydrous gel, a serum, a dispersion of vesicles, or apowder.

“Physiologically acceptable” means that the compositions are suitablefor topical or transdermal use, in contact with mucous membranes,appendages (nails, hairs), scalp and skin of mammals, particularlyhuman, compositions which may be ingested, or injected into the skin,without risk of toxicity, incompatibility, instability, allergicresponse, and others.

This “physiologically acceptable medium” forms what is commonly calledthe excipient of the composition.

The alkyl-phthalide(s) or plant extract comprising such may be combinedwith other active ingredients at effective concentrations that can actsynergistically or additionally for reinforcing and achieving thedesired effects described for the invention, such as the followingagents: UVA and/or UVB radiation filtering agents, hydrating,moisturizing, humectant, calming, dermo-relaxing, slimming,restructuring, firming, replumping, lifting, antidandruff, smoothing,acting on blood microcirculation, inflammation, free radicals,anti-aging, anti-fine lines and wrinkles, lightening, acting oncomplexion, anti-glycation, anti-carbonylation, pro-pigmenting, actingon stratum corneum, on dermal-epidermal junction, on HSP proteinproduction, on firmness, elasticity and tone of skin, on hair growth oranti-regrowth (including eyelashes and eyebrows), on eye contours (darkcircles and under eye bags), peptides, vitamins, etc.

For a scalp treatment in addition to or reinforcement of activity ispreferably used:

-   -   An anti-dandruff active acting as antifungal: such as zinc        pyrithione, ketoconazole, climbazole, piroctone olamine or        selenium disulphide;    -   A moisturizing agent such as DuraQuench™ (Croda);    -   An active rebalancing the skin microflora as HAIRSPA™ (Sederma);    -   A calming active as PACIFEEL™ (Sederma); and/or    -   An antibacterial, moisturizing and anti-stinging active as        OSMOCIDE™ (Sederma); and/or    -   A detoxification and skin reinforcement against air pollutants        active as CITYSTEM™ (Sederma); and/or    -   A cosmetic active to prevent hair loss and stimulate their        growth as CAPIGENE™, CAPILECTINE™, PROCAPIL™ (Sederma); or to        reinforce the structure of damaged hair as CERAMIDE A2™,        HELIOGENOL™ (Sederma); or to smooth the hair as FRUIT BIO™        (Sederma).

The treatment of the invention may be applied to all body parts, andmore specifically according to the preconized indication to the face,body, neckline or scalp, in whatever form or carrier known to thoseskilled in the art, in particular in the form of solution, dispersion,emulsion, paste, or powder, individually or as a premix or in vehiclesindividually or as a premix in vectors such as macro-, micro- ornano-capsules, macro-, micro- or, nano-spheres, liposomes, oleosomes orchylomicrons, macro-, micro-, or nanoparticles or macro-, micro- ornano-sponges, micro- or nano-emulsions or adsorbed on organic polymerpowders, talcs, bentonites, spores or exines, and other inorganic ororganic supports. In cosmetics in particular, applications can beoffered in skincare ranges for the face, body and scalp. In general, thealkyl-phthalide(s) or plant extract(s) according to the invention may beused in any form, in a form bound to or incorporated in or absorbed inor adsorbed on macro-, micro-, and nanoparticles, or macro-, micro-, andnano-capsules, for the treatment of textiles, natural or syntheticfibres, wools, and any materials that may be used for clothing orunderwear for day or night intended to come into contact with the skin,handkerchiefs or cloths, to exert their cosmetic effect via thisskin/textile contact and to allow continuous topical delivery.

The CTFA («International Cosmetic Ingredient Dictionary & Handbook»(16th Ed. 2016) published by «the Personal Care Products council»,ex-«the Cosmetic, Toiletry, and Fragrance Association, Inc.»,Washington, D.C.), describes a non-limited wide variety of cosmetic andpharmaceutical ingredients commonly used in the skin care industry,which are suitable for use as additional ingredients in the compositionsaccording to the present invention.

Further additional skin care actives that are particularly useful can befound in the commercial literature of Sederma and on the websitewww.sederma.com.

The following commercial actives can also be mentioned, as examples:betaine, glycerol, Actimoist Bio 2™ (Active organics), AquaCacteen™(Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia),Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (ArchChemicals, Inc), Hydra'Flow™ (Sochibo), Hydromoist L™ (Symrise),RenovHyal™ (Soliance), Seamoss™ (Biotech Marine), Argireline™(commercial name for the acetyl hexapeptide-3 of Lipotec), spilanthol oran extract of Acmella oleracea known under the commercial name GatulineExpression™, an extract of Boswellia serrata known under the commercialname Boswellin™, Deepaline PVB™ (Seppic), Syn-AKE™ (Pentapharm),Ameliox™, Bioxilift™ (Silab), PhytoCellTec™ Argan (Mibelle), PapilactylD™ (Silab), Preventhelia™ (Lipotec), and from Sederma: Subliskin™,Venuceane™, Moist 24™, Vegesome Moist 24™, Essenskin™, Juvinity™,Revidrat™, Resistem™, Chronodyn™, Kombuchka™, Chromocare™,Calmosensine™, Glycokin factor S™ Biobustyl™, Idealift™, Ceramide 2™,Ceramide A2™ et Ceramide HO3™, Legance™, Intenslim™, Prodizia™,Beautifeye™, Pacifeel™, NG-shea butter unsaponifiables (natural grade),Zingerslim™, Meiritage™, Senestem™, Sebuless™, Majestem™, Rubistem™,Citystem™, or mixture thereof.

Among other plant extracts which can be combined with the alkylphthalide(s) or plant extract(s) of the invention, there may moreparticularly be mentioned extracts of Ivy, in particular English Ivy(Hedera helix), of Bupleurum chinensis, of Bupleurum falcatum, of arnica(Arnica montana L), of rosemary (Rosmarinus officinalis N), of marigold(Calendula officinalis), of sage (Salvia officinalis L), of ginseng(Panax ginseng), of ginko biloba, of St.-John's-Wort (Hyperycumperforatum), of butcher's-broom (Ruscus aculeates L), of Europeanmeadowsweet (Filipendula ulmaria L), of big-flowered Jarva tea(Orthosiphon stamincus benth), of artichoke (Cynara scolymus), of algae(Fucus vesiculosus), of birch (Betula alba), of green tea, of cola nuts(Cola nipida), of horse-chestnut, of bamboo, of Centella asiatica, ofheather, of fucus, of willow, of mouse-ear, of escine, of cangzhu, ofchrysanthellum indicum, of the plants of the Armeniacea genus,Atractylodis platicodon, Sinnomenum, Pharbitidis, Flemingia, of Coleussuch as C. Forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C.xanthantus and C. Barbatus, such as the extract of root of Coleusbarbatus, extracts of Ballote, of Guioa, of Davallia, of Terminalia, ofBarringtonia, of Trema, of antirobia, cecropia, argania, dioscoreae suchas Dioscorea opposita or Mexican, extracts of Ammi visnaga, ofSiegesbeckia, in particular Siegesbeckia orientalis, vegetable extractsof the family of Ericaceae, in particular bilberry extracts (Vacciniumangustifollium) or Arctostaphylos uva ursi, aloe vera, plant containingsterols (e.g., phytosterol), Manjistha (extracted from plants of thegenus Rubia, particularly Rubia cordifolia), and Guggal (extracted fromplants of the genus Commiphora, particularly Commiphora mukul), kolaextract, chamomile, red clover extract, Piper methysticum extract (KavaKava™ from Sederma), Bacopa monieri extract (Bacocalmine™ from Sederma)and sea whip extract, extracts of Glycyrrhiza glabra, of mulberry, ofmelaleuca (tea tree), of Larrea divaricata, of Rabdosia rubescens, ofEuglena gracilis, of Fibraurea recisa Hirudinea, of Chaparral sorghum,of sun flower extract, of Enantia chlorantha, of Mitracarpe ofSpermacocea genus, of Buchu barosma, of Lawsonia inermis L., ofAdiantium capillus-veneris L., of Chelidonium majus, of Luffacylindrica, of Japanese Mandarin (Citrus reticulata Blanco var. unshiu),of Camelia sinensis, of Imperata cylindrica, of Glaucium flavum, ofCupressus sempervirens, of Polygonatum multiflorum, of loveyly hemsleya,of Sambucus nigra, of Phaseolus lunatus, of Centaurium, of Macrocystispyrifera, of Turnera diffusa, of Anemarrhena asphodeloides, of Portulacapilosa, of Humulus lupulus, of Coffea arabica, of Ilex paraguariensis,or of Globularia cordifolia, of Albizzia julibrissin, of Oxydendronarboretum, of Zingimber zerumbet smith, of Astragalus membranaceus, ofAtractylodes macrocephalae, of Plantago lanceolata, of Leontopodiumalpinism, of Mirabilis jalapa, of Marrubium vulgare, or of orchids.

The compositions of the present invention may include one or morepeptides, including, without limitation, di-, tri-, tetra-, penta- andhexapeptides and their derivatives. According to a particularembodiment, the concentration of the additional peptide, in thecomposition, ranges from 1×10⁻⁷% and 20%, preferably from 1×10⁻⁶% and10%, preferably between 1×10⁻⁵% and 5% by weight.

According to the present invention, the term “peptide” refers topeptides containing 10 amino acids or less, their derivatives, isomersand complexes with other species such as a metal ion (e.g. copper, zinc,manganese, magnesium, and others). The term “peptides” refers to bothnatural peptides and synthetic peptides. It also refers to compositionsthat contain peptides and which are found in nature, and/or arecommercially available.

Suitable dipeptides for use herein include but are not limited toCarnosine (βAH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM or PP.

Suitable tripeptides for use herein include, but are not limited to RKR,HGG, GKH, GGH, GHG, KFK, KAvaK, KPAK, KAbuK, KAcaK, KPK, KMOK, KMO₂K(MO₂ being a di-oxygenated sulfoxide methionine), KVK, PPL, PPR, SPR,QPA, LPA or SPA.

Suitable tetrapeptides for use herein include but are not limited toRSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2), KTFK (SEQ ID NO: 3), KTAK (SEQID NO: 4), KAYK (SEQ ID NO: 5) or KFYK (SEQ ID NO: 6).

Suitable pentapeptides include, but are not limited to KTTKS (SEQ ID NO:7). Suitable hexapeptides include but are not limited to GKTTKS (SEQ IDNO: 8) and VGVAPG (SEQ ID NO: 9).

Other suitable peptides for use herein include, but are not limited to:lipophilic derivatives of peptides, preferably palmitoyl (Pal)derivatives, and metal complexes as aforementioned (e.g. copper complexof the tripeptide HGG). Preferred dipeptide include for exampleN-Palmitoyl-β-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (Calmosensine™,Idealift™ from Sederma), Pal-RT or Pal-KT (Sederma). Preferredtripeptide derivatives include for example Pal-GKH and Pal-GHK (fromSederma), the copper derivative of HGG (Lamin™ from Sigma), Lipospondin(N-Elaidoyl-KFK) and its analogs of conservative substitution,N-Acetyl-RKR-NH₂ (Peptide CK+), N-Biot-GHK (from Sederma), Pal-KAvaK,Pal-KβAlaK, Pal-KAbuK, Pal-KAcaK, or Pal-KMO₂K (Matrixyl® synthe'6® fromSederma), PalKVK (Syn-Coll™ of DSM), and derivatives thereof.

Here can also be cited the anti-aging tripeptides of general formulaX-Pro*-Pro*-Xaa-Y disclosed in WO2015181688 with Xaa selected from Leu,Arg, Lys, Ala, Ser, and Asp; at the N terminal end, X selected from H,—CO—R₁ and —SO₂—R₁ and at the C terminal end Y selected from OH, OR₁,NH₂, NHR₁ or NR₁R₂; R₁ and R₂ being, independently from each other,selected from an alkyl, aryl, aralkyl, alkylaryl, alkoxy et aryloxygroup, that can be linear, branched, cyclic polycyclic, saturated,unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfured,said group having or not an O, S and/or N heteroatom in its skeleton andPro* corresponding to a Proline, analog or derivative thereof;comprising for example Myr-PPL-OH and Myr-PPR-OH.

Here can further be cited also the propigmenting and/or pro-mecdipeptides and tripeptides of general formula X-(Xaa₁)n-Pro*-Xaa₂-Ydisclosed in WO2014/080376, with n=0, 1 or 2, Xaa₁ an hydrophobicaminoacid selected from Ala, Val, Met, Leu, Iso, Phe, Pro, and analogsand derivatives thereof; or a polar aminoacid selected from Ser, Thr,Tyr, Asp, Glu and analogs and derivatives thereof; and when n=2 the twoaminoacids Xaa₁ being the same or different; Xaa₂ being an hydrophobicaminoacid selected from Ala, Val, Met, Leu, Iso, Phe, and analogs andderivatives thereof, or a basic aminoacid selected from Arg, Lys, His,and analogs and derivatives thereof; at the N terminal end X beingselected from H, —CO—R₁ and —SO₂—R₁; at the C terminal end Y beingselected from OH, OR₁, NH₂, NHR₁ or NR₁R₂; R₁ and R₂ being,independently from each other, selected from an alkyl, aryl, aralkyl,alkylaryl, alkoxy et aryloxy group, that can be linear, branched, cyclicpolycyclic, saturated, unsaturated, hydroxylated, carbonylated,phosphorylated and/or sulfured, said group having or not an O, S and/orN heteroatom in its skeleton and Pro* corresponding to a Proline, analogor derivative thereof; comprising for example the following peptidesPal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPAOH, Myr-SPA-OH, Pal-PM-OH,Pal-PA-OH and Pal-PP-OH.

Suitable tetrapeptide derivatives for use according to the presentinvention include, but are not limited to, Pal-GQPR (SEQ ID NO: 10)(from Sederma) and Pal-KTFK (SEQ ID NO: 11) or Ela-KTFK (SEQ ID NO: 12),Ela-KTAK (SEQ ID NO: 13), Ela-KAYK (SEQ ID NO: 14) or Ela-KFYK (SEQ IDNO: 15). Suitable pentapeptide derivatives for use herein include, butare not limited to, Pal-KTTKS (SEQ ID NO: 16) (available as Matrixyl®from Sederma), Pal-YGGFXaa (SEQ ID NO: 17) with Xaa being Leu or Pro, ormixtures thereof. Suitable hexapeptide derivatives for use hereininclude, but are not limited to, Pal-VGVAPG (SEQ ID NO: 18), Pal-GKTTKS(SEQ ID NO: 19), Pal-HLDIIXaa with Xaa being Trp, Phe, Tyr, Tic,7-hydroxy-Tic ou Tpi (SEQ ID NO: 20) and derivatives thereof. Themixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 10) (Matrixyl® 3000,Sederma) can also be mentioned.

The preferred compositions commercially available containing atripeptide or a derivative include Biopeptide-CL™, Maxilip™, Biobustyl™,Procapil™ and Matrixyl® synthe'6® of Sederma. The compositionscommercially available preferred sources of tetrapeptides includeRigin™, Eyeliss™, Matrixyl® Reloaded and Matrixyl 3000® which containbetween 50 and 500 ppm of Pal-GQPR (SEQ ID NO: 10) and an excipient,proposed by Sederma.

The following marketed peptides can be mentioned as well as additionalactive ingredients:

-   -   Vialox™ (INCI name=Pentapeptide-3 (synthetic peptide comprising        alanine, arginine, isoleucine, glycine and proline)), Syn-ake™        (β-Ala-Pro-Dab-NH-Bzl) or Syn-Coll™ (Pal-Lys-Val-Lys-OH)        marketed by Pentapharm;    -   Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH₂ (INCI name=Acetyl        hexapeptide-3) (SEQ ID NO: 21), Leuphasyl™        (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO: 22), Aldenine™        (Gly-His-Lys), Trylagen™ (INCI name=Pseudoalteromonas Ferment        Extract, Hydro lyzed Wheat Protein, Hydro lyzed Soy Protein,        Tripeptide-10 Citrulline (reaction product of Citrulline and        Tripeptide-10 (synthetic peptide constituted of aspartic acid,        isoleucine and lysine)), Tripeptide-1), Eyeseryl™        (Ac-β-Ala-His-Ser-His)(SEQ ID NO: 23), Serilesine™        (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO 24) or Decorinyl™ (INCI        name: Tripeptide-10 Citrulline=reaction product of Citrulline        and Tripeptide-10 (synthetic peptide constituted of aspartic        acid, isoleucine and lysine) marketed by Lipotec;    -   Collaxyl™ (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO 25)) or        Quintescine™ (Cys-Gly) marketed by Vincience;    -   Cytokinol™ LS (casein hydrolysate) marketed by Les Laboratoires        Serobiologiques/Cognis;    -   Kollaren™ (Gly-His-Lys), IP2000™ (Pal-Val-Tyr-Val) or Meliprene™        (INCI name=Monofluoroheptapeptide-1: reaction product of acetic        acide and a synthetic peptide comprising arginine, glycine,        glutamic acid, histidine, norleucine, p-fluorophenylalanine and        tryptophan) marketed by l'Institut Européen de Biologie        Cellulaire;    -   Neutrazen™ (Pal-His-D-Phe-Arg-NH₂) marketed by Innovations; or    -   BONT-L-Peptide™ (INCI name=Palmitoyl Hexapeptide-19: reaction        product of palmitic acid and Hexapeptide-19 (synthetic peptide        constituted of asparagine, aspartic acid, lysine and        methionine), Timp-Peptide™ (INCI name=Acetyl Hexapeptide-20:        reaction product obtained by acetylation of Hexapeptide-20        (synthetic peptide constituted of alanine, glycine, lysine,        valine and proline) or ECM Moduline™ (INCI name=Palmitoyl        Tripeptide-28: reaction product of palmitic acid and        Tripeptide-28 (synthetic peptide constituted of arginine, lysine        and phenylalanine) marketed by Infinitec Activos.

More specifically, according to the invention the alkyl-phthalide(s) orplant extract comprising such may be combined with at least one ofcompounds selected from compounds of the vitamin B3, compounds such asniacinamide or tocopherol, retinoid compounds such as retinol,hexamidine, α-lipoic acid, resveratrol or DHEA, hyaluronic acid,peptides, in particular N-acetyl-Tyr-Arg-O-hexadecyl ester, Pal-VGVAPG(SEQ ID NO: 18), Pal-KTTKS (SEQ ID NO: 16), Pal-GHK, Pal-KMO₂K andPal-GQPR (SEQ ID NO: 10), which are widely used active ingredients intopical cosmetic or dermopharmaceutical compositions.

The present invention also provides a method of cosmetic ordermatological topical treatment for improving the appearance andcondition of the skin and scalp, comprising the topical application tothe skin of a subject in need thereof an effective amount of at leastone alkyl-phthalide or a plant extract comprising in majority saidalkyl-phthalide or a composition comprising them, in a physiologicallyacceptable excipient.

“Topical treatment” or “topical use” means according to the invention,an application that is intended to act where it is applied: skin and/orscalp.

A composition according to the invention may be applied locally totargeted areas, for example using a cannula type of applicator suitablefor the scalp.

The “effective” amount depends on various factors, such as the age, thecondition of the patient, the seriousness of the disorder or pathology,the administration mode, etc. An effective amount means a non-toxicamount enough to achieve the desired effect.

All percentages and ratios used herein are by weight of the totalcomposition and all measurements are made at 25° C. unless it isotherwise specified.

For example, for a cosmetic treatment of the face, the EuropeanCosmetics Directive has set a standard amount for applying a cream of2.72 mg/cm²/day/person and for a body lotion of 0.5 mg/cm²/day/person.

For example, for a scalp hair treatment, a dose of total phthalides from0.5 to 2 mg is recommended to apply per week for at least 1 week,preferably at least 3 weeks. For example according to a treatment of atleast 3 times per week, applying a dose of 10 grams of shampoo, and then10 grams of a non-rinsed after-shampoo lotion (“Leave-on”) withrespectively 2% (shampoo) and 3% (“Leave-on”) of the active ingredientcontaining the extract according to the invention (according to formulas1.1 and 1.2 of the § D Galenics data below).

According to other specific features, the cosmetic treatment methodaccording to the invention can be combined with one or more othertreatment methods targeting the skin such as lumino-therapy, heat oraromatherapy treatments.

According to the invention, devices with several compartments or kitsmay be proposed to apply the method described above which may includefor example and non-restrictively, a first compartment containing acomposition comprising at least one alkyl-phthalide or a plant extractcomprising it as the major compound according to the invention, and in asecond compartment another active ingredient and/or excipient, thecompositions contained in the said first and second compartments in thiscase being considered to be a combination composition for simultaneous,separate or stepwise use in time, particularly in one of the treatmentmethods recited above.

The treatment method according to the invention is more particularlysuitable for a cosmetic treatment of the scalp, and in particular ofdandruff states, oily skins (treatment of the gloss and/or dilatedpores) of lines and wrinkles and loss of mechanical properties of theskin (loss of firmness in particular).

A) Example of Preparation of an Apium graveolens Seeds Extract that canbe Used in the Context of the Invention

The Apium graveolens seeds are ground to a particle size of powderaround 800 μm. This powder is then extracted with supercritical CO₂under 90 bars pressure and at 40° C. The residual water that may bepresent in the final extract is then removed if necessary (for exampleby decantation, vacuum evaporation, lyophilization). The extractionyield is around 2.5%. The extract is in the form of an oily liquid,clear to slightly opalescent, colorless to pale yellow.

The resulting extract is passed on Ultra High Performance LiquidChromatography (UHPLC) on a C18 column with as mobile phase awater/acetonitrile gradient, to dose the various phthalides.

This analysis confirms the presence of 71% of total phthalides in theextract comprising the 3-alkyl phthalides: 10% of 3-n-butylphthalide,30% of sedanolide and 60% of sedanenolide by weight relative to thetotal phthalides. The extract can be used pure or diluted.

B) Formulation of an Active Ingredient that can be Used in the Contextof the Invention

The active ingredient is a composition comprising the extract of Apiumgraveolens seeds obtained according to A) above dissolved in a matrixforming a physiologically acceptable medium. This active ingredient isparticularly intended for the cosmetics industry for the preparation ofcosmetics, creams, gels, etc. (See galenic examples at point D) below).

An extract obtained according to A) above can be diluted in anyphysiologically acceptable fatty excipient to reach at the end aconcentration of 500 ppm of alkyl-phthalides. Esterified oil ispreferably used of Caprylic/Capric Triglyceride type for this dilution.

For example, and for the description of the in vivo tests and thegalenic of point D) below, it is this dilution that has been preferablyused. This constitutes the active ingredient that will used itselfpreferably at between 1 and 5% in a cosmetic composition that can beapplied on the skin.

C) In Vitro Test Results

In vitro tests were carried out from a crude extract of seed oil ofApium graveolens manufactured according to the production methoddescribed in A) above. In general, 8, 16 and 24 ppm of this extract,respectively corresponding to 5, 10 and 15 ppm of total phthalides, weretested. These amounts correspond themselves to a dosage of applicationto the skin or scalp of 1%, 2% and 3% of the above active ingredient ofpoint B).

1. Reducing the Proliferation of Germs Involved in the Formation ofDandruff

a) Action on the Production of an Antimicrobial Natural Peptide (hBD2)

To control the perturbation of its microbial flora, scalp producesantimicrobial natural peptides (PAM) to reduce the proliferation ofgerms (bacteria, yeast) and their harmfulness. PAM therefore protectsthe scalp. One of them, the beta-defensin 2 peptide (hBD2) is producedby skin cells and acts by perforating the cell walls of bacteria oryeast.

Principle:

Human keratinocytes were grown to mid-confluence for 4 days and receivethe product according to the invention in their culture medium, themedium being changed regularly. At the end of the contact, the cellswere lysed and the levels of hBD2 are assessed by ELISA. The number ofcells is estimated in parallel with a DNA dye for quantifying cells andnormalize the data.

TABLE 1 Modulation of the production of the antimicrobial peptide hBD2in the keratinocytes in contact with the extract according to theinvention for 4 days (n = 3). hBD2; pg/mL/10⁶ cell. Variation (%)Control 205 +/− 55 Reference Extract  8 ppm 317 +/− 36 +55%; p < 0.05according to the 16 ppm 385 +/− 35 +88%; p < 0.01 invention 24 ppm 447+/− 36 +118%; p < 0.01 

No cytotoxic effect noted.

The results show that the extract, according to the invention canincrease significantly and dose-dependently the production of theantimicrobial peptide hBD2 and thus can inhibit the proliferation ofmicroorganisms involved in dandruff.

b) Action on the Production of a Microbial Receptor (TLR9)

Epithelial cells have at their external surface or on the surface oftheir endosomes receptor called Toll-Like (or TLR). These structuresplay a crucial role in innate immunity of the skin. They aim indeed tocapture the bacteria, yeast and viruses and trigger an immune response.But too abundant, they promote inflammation. Besides TLR2 and 4, wellknown and extracellular, TLR-9 is found especially in keratinocytes andis involved in the recognition of several yeast including Malasseziafurfur and Malassezia globasa, responsible for the formation ofdandruff.

Principle:

Skin explants (5 mm diameter) from 4 female donors (mean 69 years (45-90years)) are brought into contact for 3 days, in their culture medium,with a Malassezia yeast suspension. After removal of the yeastsuspension, the skins received every day, for 3 days, an after-shampoosolution not rinsed (“Leave-on”; formula 1.2 of below § D Galenics)containing an extract of Apium graveolens according the invention(corresponding to a dose of 15 ppm of total phthalides), or the placebo.After rinsing, the skins are cut and a TLR-9 marking is performed onthese sections.

TABLE 2 Modulation of the expression of TLR9 in skin explants aftercontact with Malassezia, effect of the extract according to theinvention and its placebo (n = 4; 12 photos/donor). Vari- Vari- TLR-9;AFU* ation (%) ation (%) Control 23.0 +/− 5.5 Reference 1 — Malasseziathen 25.9 +/− 6.7 +12.6%, Reference 2  

 Leave-on 

 placebo p < 0.05 Malassezia then 21.8 +/− 4.3 — −16%;  

 Leave-on 

p < 0.01 according to the invention *AFU: Arbitrary Fluorescence Unit.

Contact with Malassezia yeast increased TLR-9 labeling in the explants.Use of the extract according to the invention enabled to control theincrease (−16%) significantly compared to placebo (p<0.01). Both testsshow that the extract according to the invention disadvantages themetabolism of yeast responsible for dandruff formation and thus allowspurifying and embellishing the scalp.

2. Reducing the Sebum Production

Oily skin is associated with a too abundant sebum production by thecells called sebocytes. Too much sebum often leads to modify theproperties of the skin and scalp.

Sebum “glues” dead skin, traps bacteria and yeast (like Malasseziafurfur at the scalp level, causing dandruff) that feed on andproliferate.

Decrease of Lipid Synthesis in the Sebocyte

Principle:

Human sebocytes (26 years female, face) were seeded in their growthmedium. At confluence, the cells were putted in contact with the extractaccording to the invention for 48 hours. After removing media,monolayers were incubated with Nile Red marker of intracellular lipidswhich estimates the amount of lipids in the cells. The estimate of theviability is performed in parallel on the same layers using afluorescent dye.

TABLE 3 Modulation of lipid synthesis in sebocytes in the presence ornot of the extract according to the invention (n = 3). Lipids; AFU*/10⁶cell. Variation (%) Control  282975 +/− 11632 Reference Extractaccording to the  8 ppm 255691 +/− 7322 −10%; p < 0.05 invention 16 ppm198976 +/− 9368 −30%; p < 0.01 24 ppm 169424 +/− 433  −40%; p < 0.01*AFU: Arbitrary Fluorescence Unit; no cytotoxic effect noted. Linoleicacid: +66% of lipids

These results show that exposure of sebocytes to the extract accordingto the invention can significantly reduce and dose-dependently theamount of lipids in the cells that produce the sebum.

The extract according to the invention can be used to treat skindisorders associated with oily skin or with an oily tendancy, like shinyor glossy aspect, size and number of pores.

The scalp can be “cleaned up” with less sebum, ground for bacteria andyeasts, and traps for dandruff.

3. Reinforcement of the Skin Barrier and its Functions

The scalp of people with a dandruff state presents cells that are poorlydifferentiated, little cornified and less cohesive, presents lessprevalence of ceramide type lipids, an increased transepidermal waterloss and a disruption of hydration. Improvement of the skin barrierupstream also helps prevent a dandruff state.

a) Preliminary Study on DNA-Array

A DNA-Array study on normal human keratinocytes in contact with theextract of the invention showed the induction of several genesassociated with the reinforcing of the skin barrier compared to thecontrol.

-   1) The expression of genes coding for loricrin, involucrin and two    forms of filaggrin (filaggrin and filaggrin-2) is increased in    contact with the extract according to the invention. These proteins    are well known to be major players in the cornification and    hydration of the stratum corneum.-   2) In parallel, the increase of the expression of the cornuline gene    is also observed in contact with the extract according to the    invention. This protein, found on the scalp, is a marker of the    terminal differentiation of the epidermis. Its importance in the    homeostasis of the stratum is underlined by the fact that it is    reduced in eczema, a chronic inflammatory disease skin.-   3) Finally, the extract according to the invention increases the    production of SPINK5 and, in parallel, moderates the gene expression    of KLK6 and 13. Desquamation results from a balance between KLK    (Kallikrein-related peptidase), which break the ties of the    corneocyte between them and SPINK5 (Serine Protease Inhibitor    Kazal-type 5) that moderates their activity. Loss of function of    SPINK5 causes hyper-desquamation of immature cells and a loss of    homeostasis of the stratum corneum.

b) Improvement of the Quality of Keratinocyte Differentiation

Normal human keratinocytes almost at confluence are contacted with theextract according to the invention (8 ppm) in an appropriate culturemedium in order to study their differentiation in microscope byfollowing the aspect of the layers. At 3 days, the aspect ofdifferentiation is assessed visually. With the extract according to theinvention a marked acceleration of the differentiation is observed withstimulation of the formation of typical structures of the top layers ofthe epidermis (presence of branched structures characteristic of theprotein-lipid rigid matrix and in multilayers of the horny envelope;refractive network).

c) Protein Markers of Epidermal Differentiation

Involucrin, loricrin, filaggrin, LCE3B (“Late Cornified Envelope protein3B”) and SPRR (“small proline-rich region proteins”) are among theproteins responsible for the quality of the barrier function confered tothe stratum corneum.

Principle:

the same cell layers as those discussed above in the fresh state in partb) are fixed and immunolabeled to visualize the synthesis of these 5protein markers of epidermal differentiation. 5 photos are made on eachof the 3 replicas. A quantification of the labeling is performed byimage analysis. A counter-staining of cell nuclei can estimate thenumber of cells and thus normalize the data.

Involucrin

TABLE 4 Modulating the expression of involucrin by keratinocytes incontact with the extract according to the invention (15 photos/case)Involucrin (AFU*/10⁶ cell.) Variation Control  7.5 +/− 9.4 RéférenceExtract accrording to the  8 ppm 206 +/− 94  ×27; p < 0.01 invention 16ppm 1134 +/− 291 ×151; p < 0.01 24 ppm 1014 +/− 74  ×135; p < 0.01 *AFU:Arbitrary Fluorescence Unit; no cytotoxic effect noted.

Loricrin

TABLE 5 Modulating the expression of loricrin by keratinocytes incontact with the extract according to the invention (15 photos/case)Loricrin (AFU*/10⁶ cell.) Variation Control  39 +/− 28 Reference Extractaccording to the  8 ppm 149 +/− 76  ×3.8; p < 0.01 invention 16 ppm  491+/− 369 ×12.6; p < 0.01 24 ppm 1161 +/− 501 ×29.8; p < 0.01 *AFU:Arbitrary Fluorescence Unit; no cytotoxic effect noted.

Filaggrin

TABLE 6 Modulating the expression of filaggrin by keratinocytes incontact with the extract according to the invention (15 photos/case)Filaggrin (AFU*/10⁶ cell.) Variation Control 182 +/− 150 ReferenceExtract according to the  8 ppm 460 +/− 263 +153%; p < 0.01 invention 16ppm 613 +/− 182 +237%; p < 0.01 24 ppm 347 +/− 138  +91%; p < 0.01 *AFU:Arbitrary Fluorescence Unit; no cytotoxic effect noted.

In parallel, the production of the protein SPRR2B (SPRR marker) isincreased in the keratinocytes in culture of 103% to 139% (p<0.01) incontact of 16 to 20 ppm of the extract according to the inventioncompared to the control.

In addition, the LCE3B, induced when it is necessary to repair the skinbarrier, show an increased production of +41% to +70% (p<0.01) with 16to 24 ppm of the extract according to the invention.

All these results are consistent to show the interest of the Apiumgraveolens seed extract according to the invention in the establishment,reinforcement and restoration of the skin barrier. Protein markers knownfor the quality of the formation of this barrier: involucrine, loricrin,filaggrins, but also SPRR2B or LCE3B, are all increasing thanks to theextract according to the invention.

d) Epidermal Lipids: Cholesterol, Ceramide 2 and Neutral Free FattyAcids

The scalp of persons with dandruff is characterized by lower levels ofintercellular lipids in the horny layer (including ceramides,cholesterol and neutral lipids). This contributes to the weakening ofthe skin barrier.

Cholesterol

The DNA-Array study also showed, in the presence of the extractaccording to the invention, the induction of the CYP51A1 gene, encodinga protein of the cytochrome P450 family, which participates incholesterol synthesis by catalyzing the removal of the lanosterol14alpha-methyl group.

Neutral Lipids

A similar culture to that carried out for proteins (above) is labeledwith a specific dye of neutral lipids (cholesterol and derivatives,triglycerides and fatty acid). After photos, an image analysis canestimate the amount of neutral lipids. The amount of cells was estimatedusing a counterstain of the nuclei.

TABLE 7 Modulation of the production of neutral lipids by keratinocytesin contact with the extract according to the invention (15 photos/case)Lipids (AFU*/10⁶ cell.) Variation Control  22 +/− 11 Reference Extractaccording to the  8 ppm 132 +/− 85   ×6; p < 0.01 invention 16 ppm 233+/− 68 ×10.6; p < 0.01 24 ppm 230 +/− 71 ×10.5; p < 0.01 *AFU: ArbitraryFluorescence Unit; no cytotoxic effect noted.

Ceramides on Keratinocyte Culture and Explants

A similar culture to that performed above is labeled with an antibodyspecific of ceramide 2. After taking the photos, image analysis canestimate its amount. The amount of cells was estimated using acounterstain of the nuclei.

Moreover, the same 4 explants as those used for the TLR9 study areimmuno-marked to reveal changes in ceramide 2 content. After photos(n=12), an image analysis can quantify them.

TABLE 8 Modulation of the production of ceramics 2 by keratinocytes andexplants in contact with the extract according to the invention (15photos/case) Keratinocytes Explants Ceramide 2* Variation Ceramide 2*Variation Control/placebo 456 +/− 471 Reference 18.9 +/− 7.2  ReferenceExtract according 944 +/− 364 +107%; p < 0.01 26.4 +/− 11.2 +40%; p <0.01 to the invention** *in AFU 10⁶ cell.: Arbitrary Fluorescence Unit;no cytotoxic effect noted. **Extract according to the invention: 16 ppmfor the keratinocytes; 24 ppm in the  

 Leave-on 

 solution for the explants.

In addition to the increase of expression of an enzyme involved in theformation of cholesterol, the increase in neutral lipids and ceramide 2,components of the skin barrier, is noted. These results thus show thatcontact with the extract according to the invention strengthens thelipid composant of the skin barrier.

4. Soothing the Scalp—Reduction of Irritants Signals

a) Effect of the Extract of the Invention on the Production of IL-8

IL-8 is a mediator produced inter alia by keratinocytes in response tothe detection of microbial or chemical agents or following UV treatment.The IL-8 guides the immune cells to the origin of the irritation andhigh levels of IL-8 are found in the squams of people with dandruff.With histamine, it is one of clear markers of dandruff.

Yeast, because of their lipase, excretes irritating lipid byproducts ofunsaturated fatty acids type or substrates for the eicosanoid pathway. Amodel solution of these lipid byproducts (SPL) is prepared and used toassess the soothing properties of the extract according to theinvention.

Principle:

Normal human keratinocytes brought almost to confluence are contacted 24h with the growth medium containing or not containing the extractaccording to the invention. The media are replaced by the same mediasupplemented or not with the SPL model solution. After 24 hours, thesupernatants were assayed for their content of IL-8 and an estimate ofthe number of cells is made by the MTT method.

TABLE 9 Modulation of the production of IL-8 by keratinocytes aftercontact with SPL - Effect of the extract according to the invention (n =5). Without SPL With SPL IL-8 IL-8 (pg/10⁶ cell.) Variation (%) (pg/10⁶cell.) Variation (%) Control 1012 +/− 172 Reference 1 1639 +/− 518 +62%; Reference 2 Extract 16 ppm  597 +/− 374 −41%, p < 0.01 982 +/− 130−40%; p < 0.01 according to 20 ppm 408 +/− 88 −60%, p < 0.01 796 +/− 103−51%; p < 0.01 the invention 24 ppm 249 +/− 32 −75%, p < 0.01 569 +/−100 −65%; p < 0.01 * no cytotoxicity was observed

The SPL increase the production of IL-8 by keratinocytes (+62%; p<0.01),thus modeling what is observed in dandruff states. The extract accordingto the invention can clearly and significantly reduce the overproductionof 40 to 65% (p<0.01). Moreover, the extract of the invention alsoreduced markedly and significantly the basal production of IL-8 41 to75% (p<0.01).

5. Action on Dermal Extracellular Matrix—Stimulation of CollagenSynthesis

The extracellular matrix is the center piece of maintaining the skinvitality and viscoelastic properties of the dermis. The components ofthis matrix undergo qualitative and quantitative structural changes overtime and under the effect of free radical processes (mainly the sun,pollution and poor living conditions), leading to skin aging and theappearance of fine lines and wrinkles.

Collagen I is a crucial element for the balance of this matrix. Itsproduction decreases with age, and its fibers are degraded by endogenousand exogenous radical processes. UVs also stimulate the synthesis ofcertain proteolytic enzymes that degrade the proteins of theextracellular matrix. This results in a loss of firmness, elasticity andskin density. So collagen stimulation is a key factor in the cosmetictreatment for beautifying the skin.

Principle:

Normal human fibroblasts (NHF) are cultured for 24 h. The cells arecontacted or not with the products to be tested or their excipient atvarious concentrations for 7 days. The synthesis of type I collagenproduced by the cells in the form of extracellular matrix is thenquantified by immuno-marking on the fixed layers. A count of nucleilabeled with Hoechst is performed in parallel in order to have anestimate of the viability and to weight the data.

TABLE 10 Stimulating the synthesis of collagen I by fibroblasts, aftercontact with the extract according to the invention % de variation/Significance Product Concentration control (Student test) Extractaccording 10 ppm +388% p < 0.01 to the invention 15 ppm +404% p < 0.0120 ppm +472% p < 0.01

D) Galenic

Various cosmetic formulations are described below. Additional activeingredients, coming when appropriate in support and/or in addition tothe activity of the active ingredient according to the invention can beadded in the correct formulation part according to their hydrophobic orhydrophilic nature. These ingredients can be of any category accordingto their(s) function(s), site of application (body, face, neck, chest,hands, etc.), the desired end and the targeted consumer.

Active ingredient according to the invention used in the galenicformulations given below: CO₂ supercritic extract of Apium graveolensseeds included in an ester oil of Caprylic/Capric Triglyceride type soas to be at the end at 500 ppm of total alkyl phthalides.

This ingredient is preconized between 0.1 and 10%, preferably between 1and 5%, more preferably between 2 and 3%.

1) Scalp Treatment

1.1 Shampoo

PRODUCT % INCI NAME Part A H₂O qsp100 Water Citric acid 0.15 Citric AcidTrisodic citrate 1.20 Sodium Citrate Part B Cremophor RH60 ™ 3.00 PEG-60Hydronegated Castor Oil Ingredient according to the 2.00 invention PartC Texapon NSO UP ™ 20.00 Sodium Laureth Sulfate Crodateric CAB-30LQ-(MH) ™ 5.00 Cocamidopropyl Betaine Phenoxyethanol qs PhenoxyethanolPerfume 0.10 Fragrance Crothix Liquid LQ-(RB) ™ 3.00 PEG-150Pentaerythrityl Tetrastearate & PEG-6 Caprylic/Capric Glycerides & Aqua

Protocol:

Weigh part A and heat at 75° C. in a water bath. Mix well. Weigh part Band heat at 75° C. in a water bath. Mix well. Add part B to part A understirring. Add the ingredients of part C, one by one, in the previouspart under stirring. Mix well.

Examples of active ingredient, marketed by Sederma, that can be added tothis formulation:

-   -   CERAMIDE A2 PH™: chemical analogue of ceramide 2, a naturally        occurring molecule of the hair. Acting by strengthening the hair        structure, protecting and sheathing damaged hair, including        colored or permed hair.    -   CAPIGENE SP™: ingredient comprising three complementary actives:        homotaurine, a bacterial filtrate rich in peptides and        sulfomucopolysaccharides extract of marine origin. Acts by        slowing hair loss, stimulating the renewal and regeneration of        hair and involved in the regulation of seborrhea often        implicated in alopecia phenomena. Contributes to the health and        strength of the scalp and hair.    -   HELIOGENOL™: hydro glycolic extract of sunflower seeds titrated        in polyphenols. Naturaly comprises a free radical captor.        Protects and repairs the natural and colored hair against the        aggression of shampoos and UV rays.    -   CAPILECTINE SP™: glycoprotein with a molecular weight of about        20,000 daltons, purified from Solanum tuberosum L. which has        similar characteristics to those of lectins. Stimulates hair        vitality.

1.2 «Leave on»

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Citric acid 0.15 Citric AcidTrisodic citrate 1.20 Sodium Citrate Potassium sorbate Qs PotassiumSorbate Keltrol CG-SFT ™ 0.30 Xanthan Gum Part B Cremophor RH60 ™ 0.40PEG-60 Hydronegated Castor Oil Ingredient according to the 3.00invention Part C Ethanol 96° 5.00 Glycerin Phenoxyethanol qsPhenoxyethanol Perfume 0.10 Fragrance Part D Incroquat CTC-30 LQ-(MH) ™1.00 Cetrimonium Chloride

Protocol:

Sprinkle xanthan gum under rapid stirring into part A and let rise for30 minutes. Heat part A at 75° C. in a water bath. Weigh part B and heatat 75° C. in a water bath. Mix well. Add part C, below 35° C., mix well.Add part C in the previous part under stirring; mix well. Add part Dslowly. Mix well.

Examples of active ingredient, marketed by Sederma, that can be added tothis formulation: as previously for the shampoo, and/or

-   -   PACIFEEL™: active ingredient actif marketed by Sederma,        comprising a natural extract of the Mirabilis jalapa plant also        known as the Marvel of Peru, which alleviates cutaneous        discomfort, fades redness of sensitive and reactive skin and        strengthens and hydrates the epidermis.

1.3 Tonic Emulsion

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Citric acid 0.15 Citric AcidTrisodic citrate 1.20 Sodium Citrate Potassium sorbate qs PotassiumSorbate Part B Butylen glycol 2.00 Butylen Glycol Keltrol CG-SFT ™ 0.30Xanthan Gum Part C Cremophor RH60 ™ 3.50 PEG-60 Hydronegated Castor OilIngredient according to the 3.00 invention Part D Phenoxyethanol qsPhenoxyethanol Perfume 0.10 Fragrance Part E Incroquat CTC-30 LQ-(MH) ™2.00 Cetrimonium Chloride

Protocol:

Weigh part A. Mix under stirring. Weigh part B and homogenize. Add partB to part A under stirring. Let rise for 30 minutes. Heat part A+B at55° C. in a water bath. Weigh part C and heat at 55° C. in a water bath.Add part C into part A+B under rapid stirring. Add part E in theprevious part under gentle agitation. Mix well.

An opaque fluid emulsion is obtained.

Examples of active ingredient, marketed by Sederma, that can be added tothis formulation:

-   -   PROCAPIL™: anti-hair-loss active ingredient marketed by Sederma        (WO00/58347) that combines a vitamin matrikine (biotinyl-GHK),        apigenin (a flavonoid extracted from citrus) and oleanolic acid        (root extract from Loveyly Hemsleya).

1.4 Smoothing Serum (Cream Emulsion)

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Optasense G83 ™ 0.20Carbomer Part B Zémea ™ 5.00 Propanediol Phenoxyethanol qsPhenoxyethanol Part C Ingredient according to the 3.00 invention PemulenTR-2 ™ 0.20 Acrylates/C10-30 Alkyl Acrylate Crosspolymer Part DPotassium sorbate qs Potassium Sorbate Part E H₂O 3.00 Water NaOH 30%0.30 Sodium Hydroxyde Part F Ethanol 96 surfin 8.00 Perfume 0.10

Protocol: Part A: sprinkle carbomer in water. Let swell 30 minutes.Weigh part B and mix. Add part B and mix. Add part B to part A undernormal agitation. Weigh part C and mix. Add part C to part A+B.Homogenize under stronger stirring. Weigh part D. Extemporaneously pourpart D into part A+B+C under stirring. Neutralize with part E topH=5.60+/−0.10 under normal agitation. Weigh part F and stir. Add part Fin the previous part under normal agitation.

1.5 Mask

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Potassium sorbate qsPotassium Sorbate Part B Rejuvasoft-PA-MH ™ 6.00 Polyquaternium-91 &Cetearyl Alcohol & behentrimonium Chloride & Myristyl MyristateTerraquat BD-PA-MH ™ 1.00 Bis-(Ethyl PPG-3 Behenate) DimoniumMethosulfate & Behenamidopropyl Dimethylamine Crodacol CS90-PA-(RB) ™2.00 Cetearyl Alcohol Ingredient according to the 3.00 invention Part CGlycerin 3.00 Glycerin Phenoxyethanol qs Phenoxyethanol Part D Perfume0.10 Fragrance

Protocol:

Weigh part A. Heat at 85° C. in a water bath. Weigh part B and heat at85° C. in a water bath. Weigh part C and homogenize. Add part C to partA under stirring. Add part B to part A+C under rapid stirring for 30 s.Add part D at 35° C. under stirring.

An opaque viscous cream is obtained.

Examples of active ingredient, marketed by Sederma, that can be added tothis formulation:

-   -   HAIRSPA™: Scalp moisturisation and soothing active ingredient        comprising lactitol and xylitol in glycerin, acts on skin        microflore balance to fight against scalp discomfort (dryness,        itching, dandruff, irritations).    -   FRUITBIO™: Complex of α-hydroxy acids associated with green tea        extract. Smoothing hair cuticle.

1.6 Oil

PRODUCT % INCI NAME Part A BRB CM 56 ™ Qsp100 Cyclopentasiloxane &Cyclohexasiloxane XIAMETER PMX 200 5cs ™ 6.00 Dimethicone BRB PTM 20 ™2.00 Phenyltrimethicone Crodamol DA-LQ-(RB) ™ 10.00 Diisopropyl AdipateArlamol HD-LQ-(RB) ™ 5.00 Isohexadecane Crodamol TN-[EU]-LQ-(JP) ™ 5.00Isotridecyl isononanoate Ingredient according to the 3.00 invention PartB Phenoxyethanol qs Phenoxyethanol Perfume 0.20 Fragrance Ethanol 96°5.00 Alcohol

Protocol: Weigh part A and put under gentle stirring. Weigh part B andhomogenize. Add part B into part A under gentle stirring. Mix well.

A colorless clear oil is obtained.

1.7 Cleansing Conditioner

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Potassium sorbate QsPotassium Sorbate Incromide Oxide C-LQ-(MH) ™ 2.00 CocamidopropylamineOxide & Water Lustreplex-LQ-(MH) ™ 1.50 Polyquaternium-70 & DipropyleneGlycol Part B Crodazoquat MCC-PA-MH ™ 1.50 Behentrimonium Methosulfate &Cetearyl Alcohol & Quaternium-86 Crodacol CS-PA-(RB) ™ 3.00 CetearylAlcohol Ingredient according to the 2.00 invention Part C Glycerin 3.00Glycerin Crovol A70-LQ-(RB) ™ 1.00 PEG-60 Almond GlyceridesPhenoxyethanol qs Phenoxyethanol Part D Perfume 0.10 Fragrance

Protocol: Weigh part A and heat in a water bath at 85° C. Weigh part Band heat in a water bath at 85° C. Weigh part C and homogenize. Add partC to part A under normal agitation. Add part B to part A+C under rapidstirring. Add part D at 35° C. under stirring and mix well.

Example of active ingredient, marketed by Sederma, that can be added tothis formulation:

CERAMIDE A2 PH™

1.8 Biphasic Spray

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Potassium sorbate qsPotassium Sorbate Part B Crovol A70-LQ-(RB) ™ 1.00 PEG-60 AlmondGlycerides Phenoxyethanol qs Phenoxyethanol Part C Crodamol DA-LQ-(RB) ™24.00 Diisopropyl Adipate Crodamol TN-[EU]-LQ-(JP) ™ 3.00 IsotridecylIsononanoate Ingredient according to the 3.00 invention Perfume 0.20Fragrance

Protocol: Weigh part A and mix under slow stirring. Weigh part B andhomogenize. Add part B to part A. Mix well under rapid stirring. Weighpart C and homogenize. Add part C into part A+B under stirring.

Example of active ingredient, marketed by Sederma, that can be added tothis formulation:

-   -   FRUIT BIO™

2) Skin Treatment

Face Cream for Oily Skin and/or Antiaging

PRODUCT % INCI NAME Part A H₂O Qsp100 Water Carbopol Ultrez 10 0.25Carbomer Part B Glycerin 3.50 Glycerin Part C Brij S2 SS 0.40 Steareth-2Brij S10 SO 1.20 Steareth-10 Crodafos CES 4.00 Cetearyl Alcohol &Dicetyl Phosphate & Ceteth-10 Phosphate Vaseline oil 2.50 Mineral OilXiameter PMX-0345 2.00 Cyclopentasiloxane & Cyclohexasiloxane CrodamolOSU 7.00 Diethylhexyl Succinate Preservative qs — Part D Ingredientaccording to the 3.00 invention Part E Potassium sorbate 0.10 PotassiumSorbate Part F H₂O 4.00 Water NaOH 30% 0.40 Sodium Hydroxide Part GPerfume 0.10 Fragrance

Protocol:

Part A: Sprinkle Carbomer in water and let swell for 1 hour withoutstirring. Weigh and add part B into part A. Homogenize. Heat part A+B ina water bath at 75° C. Weigh and heat part C in a water bath at 75° C.Mix well. Weigh part D. Add part D into part C, homogenize. Add part C+Din part A+B under stirring. Extemporaneously, add part E in the emulsionunder stirring. Add part F in the emulsion. Cool under stirring. AdjustpH to 5.90+/−0.10 using part F, below 35° C. Add part G, mix well.

Examples of active ingredient, marketed by Sederma, that can be added tothis formulation:

-   -   MATRIXYL™3000: peptide-based anti-wrinkle ingredient marketed by        Sederma (WO2005/048968) comprising two matrikines Pal-GHK and        Pal-GQPR, which in synergy helps repairing skin damages caused        by aging.    -   MATRIXYL synthe'6™: peptide-based anti-wrinkle ingredient        marketed by SEDERMA which helps repair skin damage caused by        aging.    -   PACIFEEL™    -   Ac-Net™: an active sold by SEDERMA (WO2003/02828692) offering a        complete treatment of oily and acne-prone skins.    -   EVERMAT™: active marketed by SEDERMA (WO2007/029187), which        decreases the secretion of sebum and thus participates in the        treatment of oily skin.

E) In Vivo Tests

Principle

Two separate studies were conducted on a total of 106 volunteers:

-   -   Study A on a panel of 46 volunteers, mean age 38 years [21-59        years], with sensitive scalp, itching and dandruff in most        cases.    -   Study B on a panel of 60 volunteers, mean age 38 years [19-63        years], half with oily dandruff and half with dry dandruff, and        itching in almost all cases.

The evaluation of the efficacy of the extract according to the inventionwas conducted on their scalp around three major axes:

1. Improvement of dandruff states:

-   -   By a dermatologist assessment,    -   Visualization on macrophotos,    -   A quantization by confocal laser microscopy and    -   A self-assessment

2. The restoration of skin barrier:

-   -   With a measure of the TEWL using a VapoMeter,    -   Of Hydration using a DermaLab™ and Corneometer™ and    -   Of sebum rate using a Sebumeter

3. The decrease of itching:

-   -   With a dermatologist evaluation and    -   A self-assessment

Protocol

Particular Inclusion Criteria

For the 2 studies, volunteers have observed a period of “wash-out” witha neutral shampoo. Hormonal constancy during the 3 months before thetest and during the test was asked to women. During the test, moderatesun exposure, the exclusive use of hair products supplied and refrainfrom going to the hairdresser were requested.

Study Type and Duration

Studies were conducted single-blind on the whole scalp. The treatmentconsisted in use, at least 3 times per week, for 3 weeks, a shampoo andthen a non-rinsed after-shampoo lotion (or “Leave-on”) either placeboeither comprising 2% respectively (shampoo) and 3% (“Leave-on”) of theingredient comprising the extract according to the invention (accordingto formulas 1.1 and 1.2 given above in § D).

The synopsis of the study can be summarized according to the followingdiagram.

T 7 days T0 (Study B only) T 21 days Dandruff Dandruff DandruffDermatologist Dermatologist Dermatologist Macrophotos . . . MacrophotosConfocal microscopy . . . Confocal microscopy . . . Self-assessmentSelf-assessment Barrier Barrier Barrier VapoMeter ™ . . . VapoMeter ™DermaLab ™ . . . DermaLab ™ Corneometer ™ Corneometer ™ Corneometer ™Sebumeter ™ Sebumeter ™ Sebumeter ™ Itching Itching ItchingDermatologist Dermatologist Dermatologist . . . Self-assessmentSelf-assessment

Statistical studies were performed using the Student's t test or, ifneeded, a Wilcoxon or Mann-Whitney non-parametric test. To compare theeffect to T0, two-sided tests were performed on paired series. Tocompare two products, two-sided tests were performed on non-pairedseries. A Chi2 test was used for the questionnaire evaluation.

Results

1) Evaluation of Dandruff

a) Clinical Evaluation by a Dermatologist (Study B)

At each interval, a dermatologist assessed the number and size of thedandruff according to the following table:

7 Numerous and large 6 Numerous and small 5 Large 4 Small 3 Few andlarge 2 Few and smal 1 None

These results show that using the treatment with the products of theinvention leads to a clear reduction in dry and oily dandruff comparedto T0 and compared to the placebo. After 3 weeks, the scores areimproved by 61% (dry dandruff, p<0.01 vs. placebo) and 64% (oilydandruff, p<0.05 vs. placebo). Scalp condition became almost normal withfew small scales (scores of 2.0 and 1.9).

Using the placebo treatment did not lead to a significant reduction indry dandruff (score decreasing from 5.0 to 4.2) and, in the oilydandruff, to a lesser score reduction score (from 5.1 to 3.9 p<0.01)than obtained with the products of the invention.

Advantageously, efficacy was noted after just 7 days on both types ofdandruff, with −31% for oily dandruff (p<0.01 vs. T0) and −20% for drydandruff (p<0.05 vs. T0).

b) Self-Evaluation by Volunteers (Study B)

At the same time, the volunteers assessed the effects of the treatmentthemselves. The results support the dermatologist's analysis.

After 3 weeks of application:

Whether for the dry or oily dandruff, 93% of the volunteers applying thetreatment with the products of the invention saw a significantimprovement in their condition (less dandruff) compared to T0 (p<0.01).These values are also significant compared to the placebo (p<0.01andp<0.05).

They are far fewer to see an improvement with the placebo (40% for dryand 53% for oily skin; not significant in 2 cases).

A much higher proportion (73 to 80%) even saw their dandruff disappearfollowing the treatment with the products of the invention. Thedifference with the panel having applied a placebo is very clear,significant (p<0.05).

c) Evaluation on Macrophotos (Study A)

Dandruff was visualized and evaluated by eight experts on standardphotos taken using a dermoscopic camera, VivaCam® (Mavig GmbH VivaScopeSystems, Germany). Each image contains 1600×1200 pixels for a 9×7 mmmeasurement field.

The photos of the volunteers showing clearly visible and sufficientdandruff at T0 were selected for the analysis, therefore a total of 17volunteers for the product and 17 volunteers for the placebo (around 70%of the panel). The photos taken after 3 weeks were compared to thosefrom T0 and the experts were to respond to the following assertion: “thescalp shows less scale after treatment”.

In the panel having used the products according to the invention, 68% ofresponses point towards a reduction in scales was noted. The 68%response rate is significantly different from all non-positive responses(8% Do not agree +24% Neither agree nor disagree). Furthermore, asignificant difference compared to the placebo panel was noted on whichonly 35% of responses are in favor of an improvement in dandruff, theundecided being almost twice as many than for the panel having appliedthe products of the invention (46% vs. 24%).

d) Evaluation by Confocal Microscopy (Study A)

It is commonly accepted that subjects with confirmed dandruff have ahyperproliferative epidermis and a thicker albeit imperfect stratumcorneum. In order to assess the effect of the products of the inventionon this parameter, the thickness of the stratum using a confocal lasermicroscope (VivaScope® 3000; Mavig GmbH, Germany) was measured.

The laser light emitted by this device in the skin is reflecteddifferently according to the structures with which it comes into contact(keratin, melanin, collagen). A specific optical system is used toreconstitute a clear image of these structures at a chosen depth.Horizontal optical sections of the epidermis and dermis, a few micronsapart in depth, are thus obtained.

During this study, images were acquired at different depths at 2.6 μmintervals in order to reproduce the entire epidermis and the stratumcorneum in particular.

TABLE 10 Evaluation of the thickness of the stratum corneum on the scalpafter 3 weeks of application of the products according to the invention(n = 23) or placebos (n = 22) Products of the invention Placebos T0 T 3weeks T0 T 3 weeks Mean (μm) +/− 20.3 +/− 17.6 +/− 19.7 +/− 21.3 +/− Sd5.8 4.4 4.8 5.0 % variation vs T0 −13.3% 8.1% Significance vs. T0 p <0.01 p < 0.05 Maximum −47% Responders   70% Significance vs. placebos p< 0.01

Analysis of the results shows an increase of 8.1% (p<0.05) of thestratum corneum thickness after 3 weeks use of the placebos. In parallelthe use of the product of the invention causes a significant 13.3%decrease in stratum corneum thickness. This reduction is alsosignificantly different from that which is observed for the placebo(p<0.01). This data would appear to indicate an improvement in stratumcorneum quality thanks to the product of the invention, and the TEWL andhydration results below confirm this.

2) Evaluation of the Cutaneous Barrier

It is known that the quality of the cutaneous barrier of the scalp isrelated in particular to a good transepidermal water loss value (orTEWL) and a good hydration.

TEWL measurement was conducted in study A. Two hydration measurementswere conducted respectively on the panel of study A and on the panel ondry dandruff of study B.

a) TEWL Measurement by the VapoMeter™ (Study A)

Plastic stencils and anatomical landmarks were used to mark out thespecific areas on the scalp. TEWL was measured nine times (3 per site×3sites) using the VapoMeter™ (Delfin Technologies, Finland) on the samesites at T0 and after T3 weeks.

TABLE 11 Evaluation of the average TEWL on the scalp after 3 weeks ofapplication of the products according to the invention (n = 20) orplacebos (n = 22) Products of the invention Placebos T0 T3 weeks T0 T3weeks Mean 18.9 +/− 17.6 +/− 19.9 +/− 20.8 +/− (g/m²/h) +/− Sd 4.2 3.84.7 2.8 % variation −6.9% +4.5% vs.T0 Significance p < 0.07 nsd vs. T0Maximum  −36% Responders   65% Significance p < 0.05 vs. placebos nds:non significant difference

These results show that the mean TEWL value is non-significantlyincreased by +4.5% following use of the placebos. At the same time, onthe panel having used the products of the invention for 3 weeks, a 6.9%decrease in mean TEWL is noted, significant at p<0.07 vs. T0 andsignificant at p<0.05 vs. placebos. This data therefore shows thebeneficial and restructuring effect of the products according to theinvention on the scalp skin barrier.

b) Evaluation of the Scalp Hydration (Study A and B)

Scalp hydration was measured by impedance measurement using aComeometer® (C&K, Germany) for the dry dandruff panel of study B andusing a DermaLab® with pin probe (Cortex, Denmark) for the completepanel of study B. The DermaLab® is especially suitable for measuringscalp hydration due to the structure of its eight-pin probe with 6 mmpins for easy access to the scalp and which prevents water collection onthe probe.

Measurements on the Corneometer® were carried out three times on thehair line whereas the DermaLab® measurements were carried out threetimes on two separate sites on the scalp.

TABLE 12 Evaluation of the hydration of the scalp after 1 and 3 weeks ofapplication of the products according to the invention or placebos.Dermalab Corneometer Products Products according to according toPlacebos the invention Placebos the invention T0 T1s. T3s. T0 T1s T3s.T0 T3s. T0 T3s. Mean 34.3 33.6 34.2 32.3 34.3 35.1 59.8 54.9 56.5 63.3+/−Sd  1.8  1.8  2.0  2.3  2.4  2.4 23.9 24.4 26.8 19.6 % variation vs.T0 −2% −0.3% +6.2%  +8.7%  −8.2% +12% Significance vs. T0 nds nds p <0.01 p < 0.01 nds p < 0.05 Maximum  18%  19% 148% Responders 100% 100% 73% Significance vs. — p < 0.01 p < 0.01 — p < 0.05 placebosCorneometer n = 15 for the 2 cases; Dermalab: n = 20 for the productsaccording to the invention, n = 22 for the placebos

These results, from two different panels, of which one specifically dry,in two separate studies and using two methods, show that application ofthe products of the invention increases scalp hydration. On a dry scalp,from 7 days an increase of +6.2% (p<0.01 vs. placebo) and an increase of+8.7% at T3 weeks (p<0.01 vs. placebo) are observed. With the pin probemethod, the results show a 12.1% increase (p<0.05 vs. placebo). Theseresults are highly complementary, the difference possibly attributableto the panel, method or the probe.

3) Evaluation of the Sebum Level

As scalp sebum level is a key criterion in the appearance of dandruff.This parameter is also centrally in the profile of an oily skin. It wasmonitored on the oily dandruff panel of study B using the Sebumeter®(C&K, Germany) which directly measures sebaceous secretion byphotometry. The Sebumeter® probe comprises a mirror on which an opaquefilm is placed and which becomes more or less transparent depending onthe sebum adsorbed by it after repeated application to the skin. Aphotoelectric cell analyses film transparency making it possible todeduce the sebum level. The oily dandruff panel was used for this study.

TABLE 13 Evaluation of sebum content on the scalp after 1 and 3 weeks ofapplication of the products according to the invention (n = 15) orplacebos (n = 15). Products of the invention Placebos T1 T1 T3 T0 weekT3 weeks T0 week weeks Mean (μg sébum/ 182.1 +/− 132.5 +/− 119.6 +/−166.5 +/− 157.8 +/− 157.5 +/− cm²) +/− Sd 12.5 8.3 8.8 8.6 9.7 10.2 %variation vs. T0 — −27.2% −34.3% — −5.2% −5.4% Significance vs. T0 p <0.01 p < 0.01 nds nds Maximum   −47%   −61% Responders   93%  100%Significance vs. placebos p < 0.01 p < 0.01

The applying of the products of the invention provides a very clearreduction in sebum level of −27%, as from the first week, which isamplified after 3 weeks to reach −34%. These decreases are highlysignificant compared to T0 (p<0.01) and compared to the placebos(p<0.01). The condition of the scalp in the subjects with oily dandruffwas clearly improved.

4) Itching

a) Itching Evaluation by Dermatologist and Self-Evaluation (Study B)

44% of the French population declares to have a sensitive scalp, andbesides it is shown that dandruff leads to a prevalence of itching andirritation that is three times higher.

After 1 and 3 weeks use of the products of the invention by the dry andoily dandruff panels, a dermatologist assessed the frequency andintensity of itching on the basis of the volunteers' declarations andaccording to the following table:

Itching score - Frequency Itching score - Intensity 1 None None 2 Mild(few episodes per day) Mild 3 Moderate (some episodes per day) Moderate4 Remarkable (numerous and frequent Patent daily episodes)

This study shows that seven days after application of the products ofthe invention, a reduction in itching, as much on dry or oily scalp, wasobserved (around −15%, p<0.05). This reduction was amplified to reach−40% (p<0.01 vs. T0 and p<0.05 vs. placebo) after 21 days of treatment.The sensation scores after 21 days of application correspondedpractically to no itching. At the same time, application of the placebosprovided almost no changes. This analysis applies to both the frequencyand the intensity of itching.

At the same time, volunteers also assessed themselves the reduction inirritation and itching and the calming effect of the treatments. Theresults support the dermatologist's analysis.

In both panels, 100% of volunteers experienced less itching after usingthe products of the invention, with a much lower percentage after usingthe placebos (66%). The variation compared to the placebos issignificant at p<0.05 for both panels. For irritation, the results werealso in favor of the products of the invention with 93% (oily dandruffpanel) and 100% (dry dandruff panel) decrease compared to just 80% and60% for the placebos.

Finally, the volunteers mainly agreed on a lasting calming effect afterapplication of the product of the invention (80% and 93%) whereas theplacebo only induced this effect on a smaller percentage at 53% in bothpanels. For the dry dandruff panel, this variation is significant withregard to the placebo panel at p<0.05.

b) Histamine on Scalp

In addition to the results on the feelings of the volunteers, histaminein the scales has been evaluated. Histamine is an immune systemsignaling molecule also found in the skin where it acts on certainreceptors and causes pruritus (skin itching). Histamine levels two timeshigher were observed on the scalp of subjects with dandruff, thesequantities clearly reducing following an effective anti-dandrufftreatment. The decrease of the level of this substance makes it possibleto predict and monitor scalp soothing.

To test the histamine levels, adhesive strips to take samples of scalesfrom subjects with dandruff were used. These samples, which werereproducible, were taken from a line running from the forehead to thevortex, marked according to anatomical landmarks and stencils between T0and T3 weeks. Once loaded with scales, the adhesive tapes were extractedin a buffer and histamine levels tested using an ELISA kit. At the sametime, the BCA method was used to test protein levels in order tonormalize the results.

43 samples were taken (3 could not be tested) from 20 people havingapplied the placebo treatment (shampoo+leave-on lotion) for 21 days, andfrom 23 others having used the treatment with the products of theinvention (shampoo at 2%+leave-on lotion at 3% of the ingredientcomprising the extract according to the invention) with the formulas 1.1and 1.2 given in the galenic part above.

TABLE 14 Modulating the amount of histamine in dander, effect of theproducts according to the invention or placebos. T0; T3 weeks; HistamineHistamine Variation (nM/mg (nM/mg Variation vs. placebos protein)protein) vs. T0 (%) (%) Placebos 7213 +/− 5926 6311 +/− 7146 −13%; nds —Products 7485 +/− 9596 4026 +/− 4723 −46%; p < 0.01 −33%; of the p <0.05 invention

The results above show that using the scalp treatment with the productsof the invention provides a reduction of the amount of histamine foundin the scales. The decrease is important (−46% compared to T0 andsignificant at p<0.01) and significant compared to the placebos (−33%,p<0.05). The reduction of this agent, known to cause itching, confirmsthe self-evaluation and the dermatologist's data.

The invention claimed is:
 1. A cosmetic active ingredient comprising ina physiologically acceptable matrix an oily extract comprising a mixtureof alkyl-phthalides, as major compounds, prepared by CO₂ supercriticalextraction of Apium graveolens seeds, wherein the mixture ofalkyl-phthalides comprises: (i) 0-45% by weight of Sedanolide, (ii)45-90% by weight of Sedanenolide, and (iii) 0-30% by weight of3-n-butylphthalide, based on the total weight of the alkyl-phthalides.2. The ingredient according to claim 1, wherein said extract comprisesat least 50% of alkyl-phthalides.
 3. The ingredient according to claim1, wherein said alkyl-phthalide mixture comprises mainly sedanenolide.4. The ingredient according to claim 3, wherein said alkyl-phthalidemixture comprises at least 50% of sedanenolide.
 5. A cosmeticcomposition comprising at least the cosmetic active ingredient accordingto claim 1 in a physiologically acceptable excipient.
 6. The ingredientaccording to claim 1, wherein the alkyl-phthalides are in a form boundto or incorporated in or absorbed in or adsorbed on macro-, micro-, andnanoparticles, or macro-, micro-, and nano-capsules, for the treatmentof textiles, natural or synthetic fibers, wools, and any materials thatmay be used for clothing to come into contact with a skin.
 7. Theingredient according to claim 1, wherein the physiologically acceptablematrix is a hydrophobic matrix or a combination of a hydrophilic matrixwith a surfactant.
 8. The ingredient according to claim 1, wherein thephysiologically acceptable matrix forms physiologically acceptablemedium comprising hydro-alcoholic solution, a water-in-oil emulsion, anoil-in-water emulsion, a micro-emulsion, an aqueous gel, an anhydrousgel, a serum, a dispersion of vesicles, or a powder.
 9. The ingredientaccording to claim 5, wherein the physiologically acceptable excipientcomprises an aqueous, a hydro-alcoholic solution, a water-in-oilemulsion, an oil-in-water emulsion, a micro-emulsion, an aqueous gel, ananhydrous gel, a serum, a dispersion of vesicles, or a powder.
 10. Thecosmetic composition according to claim 5, wherein the cosmetic activeingredient is present in an amount of 0.1-10% by weight in thephysiologically acceptable excipient, based on the total weight of thecosmetic composition.
 11. The cosmetic composition according to claim 5,wherein the cosmetic composition is a topical composition.